Synthetic propeptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae

Abstract Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony‐stimulating factor (hG‐CSF) in S. cerevisiae, a secretion‐enhancing peptide cassette including an hIL‐1β‐derived propeptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters—N‐glycosylation, net negative charge balance, and glycine‐rich flexible linker—were investigated in batch cultures of S. cerevisiae. Using a three‐stage design involving screening, selection, and optimization, the production and secretion of hG‐CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG‐CSF produced by the optimized propeptide after the final stage increased by 190% compared to that of the original propeptide. Although hG‐CSF was used as the model protein in the current study, this strategy applies to the enhanced production of other heterologous proteins, using S. cerevisiae as the host.

affect the yield of heterologous proteins. Therefore, many studies have addressed the development of the fermentation process, strain engineering using genetic modification, and the optimization of secretory expression vector systems (Besada-Lombana & Da Silva, 2019;Hou et al., 2012;Huang et al., 2018;Liu et al., 2012;Mori et al., 2015).
Among these factors, the design and use of signal/leader peptides in vector systems is a relatively simple and highly efficient approach to overcome the issues in secretory expression and is easily applicable to other host strains (Inokuma et al., 2016;Mori et al., 2015;Wirajana et al., 2016). Secretion signals in well-known secretory proteins, such as acid phosphatase, invertase, and α-mating factor, have been investigated to improve the production yield of heterologous proteins (Lin-Cereghino et al., 2013). Human interleukin-1β (hIL-1β)-derived peptide is also a signal/leader peptide capable of inducing protein secretion. Increased secretion occurs from S. cerevisiae when it is fused to the Kluyveromyces lactis killer toxin leader peptide (Fleer et al., 1991) or the Candida albicans glucoamylase leader peptide (Choi et al., 2001;Livi et al., 1991;Song et al., 2002).
However, many heterologous proteins with signal/leader peptides are still secreted at comparatively low levels, even when the transcription/translation level of the heterologous protein is optimized for overexpression. Along with simple protein expression, the secretion of heterologous proteins also depends on many factors, such as the post-translational translocation of nascent proteins into the endoplasmic reticulum (ER), protein folding and quality control inside the ER, post-translational glycosylation in the ER and Golgi apparatus, intracellular protein trafficking and sorting, proteolytic degradation, and stress responses to the misfolding or overexpression of proteins. In particular, the process of transport from the ER to the Golgi apparatus in yeast is a rate-limiting step in the secretory pathway of heterologous proteins (Bao et al., 2018;Hou et al., 2012;Van Zyl et al., 2016). Therefore, the design and optimization of secretion signal/leader peptides for nascent protein folding and the translocation of nascent proteins from the ER to the Golgi apparatus are crucial and may increase the secretion of heterologous proteins.
Human granulocyte colony-stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage, causing the proliferation and differentiation of committed precursor cells and the activation of mature neutrophils (Vanz et al., 2008).
With the increased applications of hG-CSF, attempts have been made to produce hG-CSF using various hosts. However, recombinant hG-CSF (rhG-CSF), belonging to a group of glycoproteins, is produced in a nonglycosylated form by Escherichia coli (filgrastim/Neupogen ® ; Amgen), resulting in insoluble inclusion bodies, which require additional steps such as refolding (C. K. Kim et al., 2013;Mishra et al., 2020). rhG-CSF in a glycosylated form produced from CHO cells (lenograstim/Granocyte ® ; Chugai Pharma) is available to treat specimens without further processing (M. O. Kim et al., 2006); however, the production process is time-consuming. Using yeast as a host to produce hG-CSF can increase the rate of production, while simultaneously producing the glycosylated form. Thus, attempts to produce hG-CSF using yeast are still in progress, but the production level has not yet reached a commercial scale (Aggarwal & Mishra, 2020;Lasnik et al., 2001;Wittman et al., 2004).
To improve the production efficiency of rhG-CSF from S. cerevisiae, we developed a secretion-enhancing peptide cassette consisting of four parts: the K. lactis killer toxin secretion leader peptide, an hIL-1β-derived peptide, a peptide between the pro-and mature sequences of yeast mating factor-α, and a kex2 proteolytic cleavage site. Among them, the effect of modifying synthetic propeptides containing an hIL-1β-derived peptide and yeast mating factor-α was investigated in detail in batch cultures of S. cerevisiae. In particular, through a sequential three-stage design and optimization involved in synthesized nascent protein folding and translocation, the production and secretion of heterologous protein rhG-CSF from S. cerevisiae were significantly increased compared to the original synthetic propeptide, and protein recovery was enhanced.

| Design and construction of peptide vectors
Clones at each stage were prepared using extension and fusion polymerase chain reaction (PCR) amplification using the appropriate primers, encoding the NH 2 -SacI-killer toxin leader-propeptide-Kex2 cleavage site-hG-CSF-NheI-COOH ( Figure 1a). All clones were sequentially ligated into the SacI-NheI site of the pIL20GC (Amp+) plasmid. The GAL1-10 gene upstream activation sequence and the secretion leader sequence of the K. lactis killer toxin within the plasmid expression vector (pILPRO-GC, Figure 1b) were cloned from the YEpsec. 1-hIl plasmid vector (Baldari et al., 1987, Lee et al., 1999. Using standard recombinant DNA techniques for enzymatic manipulation of cloned DNA, plasmid expression vectors were constructed and modified, together with the stepwise sequence modification of the propeptide (Figure 2).
F I G U R E 2 Schematic illustration of the experimental procedure of cell culture, sampling, detection, and quantification of recombinant human granulocyte colony-stimulating factor (rhG-CSF) bacto-peptone, and 2% glucose), and transformed according to the Alkali-Cation Yeast Transformation Kit manual (BIO 101 Inc.).
The transformants were selected on SD-Ura medium (0.8 g/L Complete Supplement Medium-URA [MP Biomedical], 6.7 g/L yeast nitrogen base without amino acids [DIFCO: Becton Dickinson and Company], 2% glucose) agar plates after incubation for 3 days at 30°C.

| Media and culture conditions
The transformed cells were cultivated overnight in a 3 ml SD-Ura liquid medium (30°C, 250 rpm). The culture broth (1 ml) was inoculated in a 50 ml SD-Ura liquid medium, followed by cultivation for 12 h at 30°C, and 210 rpm. The culture broth was centrifuged for 5 min at 2543g, and the cell pellet was resuspended in 25 ml YPGal medium (1% yeast extract, 2% bacto-peptone, and 2% galactose) to induce recombinant gene expression using the GAL1-10 upstream activating sequence, followed by a 24 h culture at 30°C.

| Extraction of recombinant protein from S. cerevisiae
The yeast culture medium was monitored until the optical density of the culture at 600 nm (OD 600 ) reached 30-40; further, the medium was diluted to 1 ml by adjusting the minimum OD to 30-40 for each stage, followed by centrifugation at 2390g for 10 min. The cell-free supernatant was condensed fivefold using a vacuum concentrator

| Analysis of secreted hG-CSF
The recombinant hG-CSF protein and hG-CSF standard (Sigma) were subjected to 4%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4%-12% Bis-Tris gel, Novex; Thermo Fisher Scientific Inc.), and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skimmed milk in TBST buffer (Trisbuffered saline [50 mM Tris-HCl, pH 7.6, and 150 mM NaCl] with 1 mM Tween 20) at 25℃ for 1-2 h, followed by incubation with the primary antibody, mouse anti-hG-CSF immunoglobin G (IgG) (Santa Cruz Biotechnology) at 4°C for 16 h. The membrane was washed twice, for 10 min each, with TBST buffer and incubated with horseradish peroxidase-conjugated antimouse secondary IgG (Santa Cruz Biotechnology) in TBST buffer for 1-2 h. After pouring off the TBST buffer, the membrane was reacted with an enhanced chemiluminescence solution (Thermo Fisher Scientific). The chemiluminescent bands were visualized using the Biomolecular Imaging System (LAS 3000 M; FujiFilm), and images were captured and analyzed using the image-processing software Multi Gauge v2.3 (Fuji Film).
A standard curve in the range of 150-700 ng was prepared using a commercial hG-CSF standard (Sigma), and rhG-CSF was quantified by the chemiluminescent method ( Figure A1). The immunoreactive protein bands were quantified through extrapolation based on the band intensity of the hG-CSF standard (200 ng) loaded on each gel.
The experimental procedure from culture to western blot analysis is shown schematically in Figure 2.
Samples treated with Endo H were confirmed by immunoblotting using a colorimetric method.

| Calculations of the % increased efficiency of secretion
The % increased efficiency of secretion of the designed propeptides in each stage was calculated using the following equation: The % increased efficiency of secretion Leu-Asp (GVSLD), which is originally located between the proand mature sequences of yeast mating factor-α, is known to enhance the efficiency of the Kex2 protease during cleavage of the C-terminal side of NH 2 -KR-COOH (Rockwell et al., 2002). In summary, hG-CSF was conjugated with the secretion-enhancing peptide cassette, which consisted of the K. lactis killer toxin secretion leader peptide, followed by a 29-residue propeptide (N-terminal 24-residue peptide [Ser5-Ala28] of hIL-1β and GVSLD) and a Kex2 proteolytic cleavage site.
The process of transport from the ER to the Golgi apparatus in yeast is a rate-limiting step in the secretory pathway of heterologous proteins (Bao et al., 2018;Hou et al., 2012;Van Zyl et al., 2016). Therefore, the secretion cassette was further designed and optimized to increase hG-CSF folding in the ER, and consequently improve the efficiency of transport into the Golgi apparatus. The enhanced folding and transition of rhG-CSF from the ER to the Golgi apparatus may significantly promote the secretion of rhG-CSF into the extracellular medium.
It has been hypothesized that the secretion of rhG-CSF would be enhanced by N-glycosylation, net charge balance, and insertion of a glycine-rich flexible linker between hIL-1β-derived peptide and hG-CSF ( Figure 3). The effects of these three factors were investigated using a three-stage screening and selection process.
The concentrations of intracellular and extracellular rhG-CSF were calculated using immunoblotting analysis at each stage of the propeptide sequence modification. Finally, the % increased efficiency of rhG-CSF secretion was estimated as the ratio of the concentration of mature rhG-CSF from the best propeptide sample in each stage to the concentration of mature rhG-CSF from the original propeptide.
3.2 | First stage design of the propeptide sequence: Inserting the N-glycosylation site N-glycosylation of the propeptide derived from hIL-1β, which occurs at a specific "Asn" site, has been found to play an important role in the secretion of heterologous proteins (K. S. Han et al., 2005;M. Han et al., 2014). N-glycosylation plays a significant role in the secretion F I G U R E 3 Propeptide sequences designed in the (a) first stage, (b) second stage, and (c) third stage (b, native sequence of original propeptide). The best propeptide sequence in the previous step is highlighted in yellow. of hG-CSF (K. S. Han et al., 2005), presumably because of the enhanced folding of rhG-CSF in the ER (Doyon et al., 2002;Sagt et al., 2000), and is subsequently involved in the transition of rhG-CSF from the ER to the Golgi apparatus (Ari et al., 2001). Therefore, we inserted up to three N-glycosylation sites by replacing Lys7 with Asn7, Gln11 with Asn11, and Val15 with Asn15 to produce Asn-X-Thr/Ser. As shown in Figures 3a et al. (1993) and Kajava et al. (2000) reported that the charge balance between the N-terminus of the signal peptide and the mature moiety is important for the secretion efficiency of gram-negative bacteria (Johansson et al., 1993;Kajava et al., 2000). In addition, acidic and neutral propeptides are known to be effective in enhancing the secretion efficiency of heterologous proteins in gram-positive bacteria (Le Loir et al., 2001). Similar research has been conducted on yeast, but the exact causal relationship has not yet been elucidated (Liang et al., 2013;Massahi & Çalık, 2016;Yarimizu et al., 2015).
In this study, all the propeptides used in the first stage had a net positive charge. Therefore, we investigated whether replacing the net positive charge with a net negative or neutral charge in the propeptide could improve the secretion efficiency. Especially, given that the aspartic acid of the protein affects solubility (Trevino et al., 2007), we replaced lysine at sites 12 and 23 in the BN3-N7,15 propeptide, with aspartic acids (Figure 3b native three-dimensional structures and become functionally independent ( Figure A2c). Figures 6 and A5, both extracellular hyper-Nglycosylated (within the blue dotted box) and mature rhG-CSF were detected by western blot analysis. The intracellular proteins also showed core N-glycosylation (within the purple dotted box) and mature rhG-CSF. After Endo H treatment, the hyper-N-glycosylated and core N-glycosylated hG-CSFs were completely converted to pro-hG-CSF, as shown in the red dotted box.

As shown in
In the third stage, except for the propeptide with the -GS linker, the other propeptides with linkers did not show a dramatic enhancement in secretion, compared to the best second-stage F I G U R E 5 (A) Representative image of western blot analyses of extracellular (a, b) and intracellular (c, d) samples from Saccharomyces cerevisiae cultures that express recombinant human granulocyte colony-stimulating factor (rhG-CSF) using the propeptides designed in the second stage. Western blot results of Endo H-treated extracellular and intracellular samples are also shown in b and d, respectively (Standard, the commercial standard of human granulocyte colony-stimulating factor [hG-CSF]; Control, samples from the culture of S. cerevisiae Y2805; B, original propeptide; BN3-N7,15, best propeptide sequence in the first stage; BN3-N7,15-K12D to BN3-N7,15-K12,23D, BN3-N7,15-derived propeptides designed in the second stage). (B) The chemiluminescent bands (extracellular mature rhG-CSF) were quantified through extrapolation based on the band intensity of the standard. Each bar represents the average with the error bars indicating standard deviations (n = 3). The p-values were analyzed by Student's t-test (*p < 0.05).
pro-peptides. BN3-N7,15-K23D with a GS linker showed the highest secretion efficiency for extracellular mature hG-CSF. Consequently, through a sequential three-stage design screening, selection, and optimization, using BN3-N7,15-K23D-GS, the amount of extracellular mature rhG-CSF increased from 28.427 mg/L (before the first stage) to 54.106 mg/L (after the third stage) in a 25-ml flask culture, and the % increased efficiency of secretion was 190% higher than that of the initial sequence of propeptide "B" (Table 1).
F I G U R E 6 (A) Representative image of western blot analyses of extracellular (a, b) and intracellular (c, d) samples from Saccharomyces cerevisiae cultures that express recombinant human granulocyte colony-stimulating factor (rhG-CSF) using the propeptides designed in the third stage. Western blot results of Endo H-treated extracellular and intracellular samples are also shown in b and d, respectively (Standard, the commercial standard of human granulocyte colony-stimulating factor [hG-CSF]; Control, samples from the culture of S. cerevisiae Y280; B, original propeptide; BN3-N7,15, best propeptide sequence in the first stage; BN3-N7,15-K23D, best propeptide sequence in the second stage; BN3-N7,15-K23D-GS to BN3-N7,15-K23D-(GSG) 2 ; BN3-N7,15-K23D-derived propeptides designed in the third stage). (B) The chemiluminescent bands (extracellular mature rhG-CSF) were quantified through extrapolation based on the band intensity of the standard. Each bar represents the average with the error bars indicating standard deviations (n = 3). The p-values were analyzed by Student's t-test (**p < 0.001).

| DISCUSSION
Heterologous protein production using S. cerevisiae has many advantages, including the secretion of proteins with post-translational processing. Although large-scale production and downstream processing of some proteins expressed in S. cerevisiae are well-established procedures, the secretion efficiency of S. cerevisiae and the conditions necessary for heterologous protein production are sometimes highly specific to the target protein and the expression system. Therefore, the system should be tailored for high-level production.
The design and optimization of signal/leader peptides is a relatively simple and efficient strategy that can be easily tuned for the high-level production of many target proteins. The hIL-1βderived peptide (Ser5 to Ala28) fused to the N-terminus of target proteins such as cytokines, hormones, enzymes, or viral surface antigens, in conjunction with an N-terminal secretion signal, improves the secretion of target proteins by S. cerevisiae (Choi et al., 2001;Song et al., 2002). The reason for the improved secretion of hIL-1β-derived peptide-linked heterologous recombinant proteins is not clear. The transport of proteins from the ER to the Golgi apparatus is a rate-limiting step that affects the secretion efficiency in yeast. The ER is equipped with relevant factors for proper protein folding (e.g., molecular chaperones-BiP, calnexin, and calreticulin and foldase-protein disulfide isomerase) and the removal of misfolded proteins (e.g., ER-associated degradation) (Brodsky & Skach, 2011;Celik & Calik, 2012). However, because these factors are also present in limited quantities, they are insufficient to accommodate all misfolded proteins. Eventually, misfolded proteins accumulate in the ER, preventing further movement along the secretory pathway. Thus, we presumed that the peptide plays an important role in enhancing target protein solubility and folding in the ER, and further in target transport to the Golgi apparatus.
However, as linking the hIL-1β propeptide does not always assure identically superior secretion efficiencies of many target proteins, a rational strategy is required to find the optimal propeptide for maximally enhancing the target protein folding and translocation for secretion of a target protein.
In this study, the strategy for heterologous protein secretory expression focuses mainly on propeptide engineering to enhance the solubility of heterologous proteins, and consequently improve the efficiency of their transport from the ER to the Golgi apparatus. in the best first-stage propeptide sequence was changed, hG-CSF secretion was almost completely inhibited. This shows that depending on the site of N-glycosylation, it may have interfered with folding and secretion (Ellgaard & Helenius, 2003;Sagt et al., 2000).
In the second stage, given that the net negative charge of the propeptide affects protein solubility (Trevino et al., 2007), the propeptide associated with hG-CSF was designed with a net negative charge and contributed to an increase in the synthesis of total (pro-and mature) rhG-CSF. However, the secretion efficiency from S. cerevisiae did not increase drastically, unlike the high secretion efficiency observed in prokaryotic expression systems, such as E. coli, indicating that secretion in eukaryotes was not significantly affected in this experiment.
In the third stage, the propeptide design incorporated a flexible linker sequence to maximize the opportunity for rhG-CSF and the propeptide derived from IL-1β to fold into their native threedimensional structures. The amount of extracellular mature rhG-CSF increased considerably when conformational flexibility was added to the propeptide by inserting internal glycine/serine residues.
When the flexible GS linker was inserted into the propeptide, the expression of extracellular mature rhG-CSF reached a maximum of 54.1 mg/L, which was 1.9-fold greater than the initial propeptide "B" (Table 1). This value is higher than that reported in other studies (yields up to approximately 39 mg/L) in which rhG-CSF was produced using the α-MAT secretion signal in another yeast species, Pichia pastoris (Aggarwal & Mishra, 2020;Lasnik et al., 2001).
T A B L E 1 The % increased efficiency of secretion of mature recombinant human granulocyte colony-stimulating factor (rhG-CSF) using the best propeptides at each optimization stage In summary, through a three-stage design involving screening, selection, and optimization, the production and secretion of rhG-SCF from S. cerevisiae substantially increased compared to the original propeptide, resulting in an enhanced protein recovery yield. Although rhG-CSF was used as the model protein in this study, this stepwise rational design of a propeptide is a promising strategy for enhancing the secretion of other heterologous proteins, using S. cerevisiae as the host.

This study was supported by grants from the National Research
Foundation of Korea (NRF) funded by the Korean government (2021R1C1C1008217 and 2020M3D3A1A01080557).

CONFLICT OF INTEREST
None declared.

DATA AVAILABILITY STATEMENT
All data are provided in full in the results section of the paper. The DNA sequence encompassing the human granulocyte colonystimulating factor (hG-CSF) is available at www.ncbi.nlm.nih.gov/ nuccore/NM_172219

ETHICS STATEMENT
Work with recombinant DNA and microorganisms was performed according to national requirements.

F I G U R E A4
(a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result of extracellular recombinant human granulocyte colony-stimulating factor (rhG-CSF) from the second stage, stained with Coomassie Brilliant Blue. The red arrow indicates mature rhG-CSF (18.6 kDa). (b) The chemiluminescent visualization of western blot analysis results of extracellular mature rhG-CSF from the second stage. (c) The chemiluminescent bands (intracellular soluble rhG-CSF) were quantified through extrapolation based on the band intensity of the standard.